CD107a expression did not necessarily correlate with expression of TNF- or IFN-. were more prominent cytokine and chemokine producers than CD56brightNK cells. The present data demonstrate how specific target cell ligands dictate qualitative and temporal aspects of NK-cell cytokine and chemokine responses. Conceptually, the results point to CD56dimNK cells as an important source of cytokines and chemokines upon recognition of aberrant cells, producing graded responses depending on the multiplicity of activating receptors engaged. == Introduction == Natural killer (NK) cells respond directly to infected or neoplastic cells through engagement of a multitude of germline-encoded receptors by ligands on target cells.13Beside their ability to kill aberrant cells, NK cells are also critical components of the innate immune response by virtue of their capacity to produce a variety of cytokines and chemokines.46Murine models have demonstrated a dependence on NK cellderived cytokines in early responses to obligate intracellular parasites such asListeria, Toxoplasma, andLeishmaniaand in resistance to cytomegalovirus infection.710In many of these systems, NK cells respond to cues from sentinel immune cells, including dendritic cells, macrophages, and pathogen-infected tissue cells.1113These cues are communicated by release of cytokines, including interleukin-1 (IL-1), IL-10, IL-12, IL-15, and IL-18.14Thus, secondary to triggering of innate immune cells by pattern recognition receptors, NK cells can relay and amplify cytokine signals. Among the most prominent cytokines produced by NK cells are tumor necrosis factor- (TNF-) and interferon (IFN-). Moreover, NK cells have been reported to secrete several other factors, including immunoregulatory cytokines such as IL-5, IL-10, IL-13, the Sarolaner growth factor GM-CSF, and the chemokines MIP-1, MIP-1, IL-8, and RANTES.1522 In humans, NK cells are usually defined as CD3CD56+cells,23and can be further subdivided based on CD56 expression. Typically, CD56dimNK cells constitute the majority (90%) of peripheral blood NK cells, whereas CD56brightNK cells are more abundant in secondary lymphoid tissues.14,24CD56dimNK cells express high levels of the low-affinity Fc receptor CD16, display variegated expression of several types of inhibitory receptors for MHC class I, and express high levels of perforin. In contrast, CD56brightNK cells express Rabbit Polyclonal to LRP11 no or low levels of CD16, exclusively express the inhibitory receptor CD94/NKG2A, and have 10-fold lower perforin expression than CD56dimNK cells.2527Because of these and other findings, CD56dimand CD56brightNK-cell subsets are considered to be developmentally distinct and to occupy different functional niches.12,2830 Human NK-cell responses to exogenous Sarolaner cytokines have been extensively studied.21In contrast, relatively less is known with respect to NK-cell cytokine and chemokine production upon target cell recognition. For example, the full spectrum of cytokines released by freshly isolated, resting NK cells upon target cell recognition has not been fully characterized. Furthermore, the minimal requirements for induction of cytokine secretion upon Sarolaner engagement of specific ligands on target cells are not known. To understand how NK cells may contribute to, and maybe even act as primary initiators of, immune responses upon target cell recognition, studies on how receptor-ligand interactions dictate qualitative and temporal aspects of cytokine Sarolaner and chemokine secretion are important. Here, we have set out to study in detail cytokine and chemokine production by human peripheral blood NK cells upon target cell recognition. To overcome the complexity in receptor-ligand interactions between NK cells and target cells, we have developed a reconstitution system usingDrosophilacells as targets. 31A notable advantage of such a system is that cytokine and chemokine secretion by primary, unmanipulated NK cells can be studied in the context of specific receptor-ligand interactions. This system has recently revealed cooperation among NK-cell receptors for discrete events in cytotoxicity, including NK-cell cytolytic granule polarization and exocytosis.32,33 Here, we addressed how specific engagement of the receptors NKG2D (CD314), DNAM-1 (CD226), 2B4 (CD244), LFA-1 (CD11a/CD18), and CD16, or combinations thereof, regulate cytokine and chemokine production by freshly isolated, resting human NK cells. The data provide insight into the regulation of cytokine and chemokine secretion by different NK-cell subsets upon target cell recognition. == Methods == == Cells == Human NK cells were isolated from peripheral blood by negative selection (NK-cell isolation kit; Miltenyi Biotec). Approval was obtained from the Regional Ethics Review Board for the use of peripheral blood mononuclear cells from healthy donors. CD56dimand CD56brightNK-cell subsets were sorted based on CD56 expression by flow cytometry (FACSAria; BD Biosciences). Freshly isolated NK cells were maintained in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum and 2mMl-glutamine; all from Invitrogen). NK-cell populations contained at least 99% CD3CD56+cells and were used within 2 days of isolation. The cell line K562 (ATCC) was maintained in complete medium. The transfection and maintenance ofDrosophilaS2 cells has been previously described and expression of the ligands for NK receptors.